Keratin-based products and methods for their productions

ABSTRACT

The present invention relates to a process for solubilizing keratins from a keratin containing starting material such as poultry feathers. The keratins are may be solubilized using a sulphide under alkaline conditions. In the process the cysteine residues of solubilized keratins and are partially modified, e.g. by alkylation. The conditions of solubilisation and partial modification are chosen such that the keratins are also partially hydrolyzed. The partially modified and partially hydrolyzed keratin may be used as stable dispersions e.g. for the production of films and coatings by casting.

FIELD OF THE INVENTION

The present invention relates to new products derived from keratins. Inparticular, the invention relates to products that are derived fromnaturally occurring sources of keratin and keratin fibres, such aspoultry feathers. The invention also relates to methods for thepreparation of such keratin-derived products and to uses of suchkeratin-derived products.

BACKGROUND OF THE INVENTION

Feathers are an important waste product of the poultry industry, withabout 4 million tons being produced per year world-wide. Although minoramounts thereof find use in for instance clothing, insulation andbedding, as well as a larger amount in the preparation of feather mealfor the production of animal feed, there are currently insufficient(economically interesting) applications for such large quantities offeathers. As for environmental reasons, burning or burying of feathersis not always a practical alternative; these amounts of waste featherspresent a difficult disposal problem for industries such as the poultryindustry.

Thus, it is a general object of the invention to provide a range ofeconomically viable uses of waste feathers, as well as to provideprocesses via which waste feathers can be put to such uses.

Feathers mainly consist of a fibrous protein material called keratin.Keratins are water-insoluble and protease-resistant proteins with amolecular weight of approximately 10 kDa. As shown in FIG. 1, eachkeratin molecule—indicated as (A) in FIG. 1—consists of a central part(β) which forms a crystalline β-sheet, and two randomly ordered chainends, indicated as (N) and (C) respectively. In the feather, the centralparts (β) of multiple keratins are joined to form a so-calledmicrofibril (B) of about 30 angstroms in diameter. The chain ends (C)and (N) contain inter- and intra-chain cross-links (C) in the form ofdisulphide bonded dimeric amino acids (cystine) and form the amorphousmatrix in which the microfibril is embedded. The disulphide bonds (C)and the molecular organisation of the proteins in the feathers impartinsolubility and resistance to most proteolytic enzymes.

There is already an extensive amount of prior art relating to keratins,keratin-hydrolysates and products derived from keratins, as well as tothe preparation and uses thereof. Especially the cosmetic and textileindustries are interested in keratin-derived products for styling andmodifying hair and wool. Shampoos and nail polish with hydrolysedkeratin are just examples of prior art formulations that make use ofkeratins. Even though these applications are numerous, they arebasically product formulations using mostly hydrolysed keratin as one ofthe components. Such products have a low added value and are of onlylimited use.

Prior art where keratins are used as a polymer in films or coatings ismuch less abundant as is evident from a review of this art in the thesisof one of the inventors (P. Schrooyen: “Feather keratins: modificationand film formation”, 1999, Thesis University of Twente, Enschede, TheNetherlands). Generally, according to the processes described in thesereferences, the insoluble keratins are extracted from their naturalsource and solubilised, e.g. in an aqueous medium. Usually, suchextraction/solubilisation involves at least disruption of the disulphidebonds (C), which breaks up the microfibrils (B) to provide theseparate(d) keratin molecules (A). Depending upon the conditions used,the extraction/solubilisation may also involve hydrolysis/degradation ofthe keratin molecules (A) themselves, i.e. cleavage of the peptide bondsbetween the amino acids that form the keratin molecule(s) (A).Disruption of disulphide bonds has reportedly been achieved by oxidationof the disulphide bonds with organic peracids to form sulphonic acidgroups; by sulphitolysis of the disulphide bonds to form S-sulphonategroups; or by reduction of the disulphide bonds with thiol compoundssuch as 2-mercaptoethanol, dithiothreitol (DTT) or dithioerytritol, orby treatment with alkali metal sulphides such as a sodium sulphidesolution. Generally, the purpose of the prior art processes is toprovide a keratin-derived product that is soluble in aqueous media, e.g.a solution of keratin(s) or keratin hydrolysate. For this purpose, theart describes modification of the disrupted disulphide bonds so as toavoid reformation of the bonds and/or the use of specific additives tokeep the keratins in solution or to otherwise stabilise the keratinsolutions, such as alkali metal hydroxides, urea, guanidinehydrochloride, 2-mercaptoethanol or thioglycolate.

E.g. U.S. Pat. No. 3,464,825 describes a process in which keratins areextracted from feathers using an alkali metal sulphide solution, such asa Na₂S-solution. The keratin-solution thus obtained is then treated withan alkali metal sulphite such as a Na₂SO₃, after which the protein isacid-precipitated. The precipitated keratin is then solubilised in aaqueous alkali and subsequently oxidised using a water soluble oxidisingagent, e.g. hydrogen peroxide, sodium periodate, sodium chlorite or anorganic peracid such as peracetic acid or performic acid, which isbelieved to oxidise the cystine/cysteine residues to cysteic acidgroups, However, the thus obtained keratins are essentially completelymodified, i.e. all free cystine/cysteine residues are oxidised tocysteic acid groups. As a result the completely modified keratins aregenerally water-soluble and, therefore, less suited to providewater-insoluble films, e.g. for applications in coatings and for otherapplications mentioned hereinbelow. Also FR 2 522 657 describesessentially complete modification, e.g. of at least 70% of the free —SHgroups of the keratins.

In contrast, U.S. Pat. No. 3,642,498, describes a process for thesolubilisation of keratins using sodium sulphide whereby thecystine/cysteine groups remain essentially unmodified. The keratins areagain extracted using an a metal sulphide solution, treated with analkali metal sulphite solution, and then acid-precipitated. Theresulting protein product is described as being dispersible inwater-alcohol mixtures, and can be used for preparing films andcoatings. U.S. Pat. No. 3,642,498 also describes alternative processesfor extracting and solubilising feather keratins, including treatmentwith mercaptoethanol-alcohol-water mixtures; or treatment with alkalinemercaptoethanol-alcohol-water mixtures containing alkali metalhydroxides. However, according to U.S. Pat. No. 3,642,498, thecystine/cysteine groups remain essentially unmodified. A majordisadvantage of solubilised keratins with essentially unmodifiedcystine/cysteine is that they do not allow to produce keratin-basedproducts, in particular films and coatings, with the desired mechanicalproperties. In particular, such films suffer from brittleness.

Most of the art mentioned thus far dates back to the late 1960's andearly 1970's. Nevertheless, in the 30 years since, these references havenot led to any widespread use of the keratin-derived products disclosedtherein. This is probably because the products and processes describedare not economically viable—despite the fact that they employ wastefeathers as a starting material—and/or because the keratin productsobtained do not show the properties required for practical (e.g.commercial) use.

Some of these problems are addressed in the thesis of one of theinventors (P. Schrooyen; “Feather keratins: modification and filmformation”, 1999, Thesis University of Twente, Enschede, TheNetherlands) that describes keratin-derived products obtained by partialmodification (i.e., alkylation) of intact feather keratins usingmonoiodoacetamide, monoiodoacetic acid or monobromosuccinic acid inconcentrated aqueous urea solution and in the presence of2-mercaptoethanol. The keratins were modified to degrees of modificationvarying between 25 and 87%, calculated on the basis of the amount ofremaining free —SH groups. This partial modification provided stabledispersions of essentially intact (i.e. non-hydrolysed), partiallymodified keratins, which could be used to cast strong films with desiredthermal and mechanical properties. However, extraction andsolubilisation of essentially intact keratins for partial modificationrequires the use of high concentrations of chemicals such as urea and2-mercaptoethanol. The use of these chemicals at experimental scale isacceptable. However, their use at large scale is not economicallyfeasible because the use of these chemicals is expensive, also in viewof the environmental and occupational hazards associated with the use ofthese chemicals, requiring expensive precautionary measures.

Thus, there is still a need for an economically viable method for theprocessing of keratin-containing (waste) materials such as feathers,which method can be used to provide a range of keratin-based products,in particular films and coatings, with properties acceptable forpractical/commercial application(s).

DESCRIPTION OF THE INVENTION

It has now been found that improved keratin-based products can beobtained by a process which involves a combination of partialdegradation, usually by means of hydrolysis, of the keratin moleculesand partial modification of the free —SH groups, i.e. the free —SHgroups resulting from cleavage of the disulphide bonds (C). Inparticular, the invention provides such a partially degraded andpartially modified product that is dispersible in water and that can beused in a range of applications, including but not limited to thosediscussed hereinbelow. Such water-dispersible, partially degraded andpartially modified keratin-based products have not yet been described inthe art.

Thus, in a first aspect, the invention relates to a process forproducing partially modified and partially degraded keratin. The processcomprises the steps of (a) solubilising keratin from a keratin-fibrecontaining starting material in an aqueous solution using a reducingagent at alkaline pH; and (b) partially modifying the —SH groups of thesolubilised keratin. Preferably the conditions of steps (a) and/or (b)are such that the solubilised keratin is partially hydrolysed orpartially degraded to a degree further specified hereinbelow. Optionallya further step (c) may be used to hydrolyse the keratin to a desireddegree.

For the keratin-fibre containing starting material any suitable sourceof keratin fibres may be used. In particular, natural sources of keratinfibres may be used, such as e.g. hair, feathers, hoofs, nails, horns andthe like. Preferably, a source of keratin fibres containing at leastβ-keratin is used. Feathers are an especially preferred startingmaterial, in particular feathers of chicken, turkey, ducks, geese orother poultry, e.g. as obtained as a waste product from the poultryindustry. These sources of keratin fibre may contain minor amounts ofother proteins and/or other components such as fat or blood, e.g.usually amounts of less than 5% of total weight (based on dry feathers).Generally, the presence thereof may be tolerated, otherwise, some or allof these non-keratin components may be removed, prior to thesolubilisation in step (a). The keratin-fibre containing statingmaterial is preferably subjected to one or more pretreatments such ase.g. cleaning, washing, sorting, defatting, cutting, milling, grinding,drying, or any combination thereof. Such pretreatment may facilitate thehandling of the starting material, it may improve the efficiency offurther processing steps, such as solubilisation of the keratin, and/orit way improve the quality of the final keratin-based product.Alternatively, an already pre-processed keratin-containing startingmaterial may be used, such as feather meal; as well as already isolatedkeratins or keratin fibres (which however for economic reasons willusually be less preferred). It is also encompassed in the scope of theinvention to use a natural source of keratin or keratin fibres—e.g.feathers—directly in the solubilisation step a), without any furtherpre-treatment.

The reducing agent for solubilisation at alkaline pH may be chosen fromsulphides, thiols, boric hydride and phosphines, or combinationsthereof. Preferred sulphides are alkali metal sulphides, such as sodiumsulphide. At lower alkaline pH, e.g. at a pH lower than 10, 9.5 or 9.0,ammonium sulphide may also be used as reducing agent for solubilisation,the use of which allows to avoid a salt residue in the final product.Preferred thiols are dithiothreitol, 2-mercaptoethanol and thioglycolateand a preferred phosphine is tri-n-butylphosphine.

The conditions of solubilisation, i.e. the concentrations of thekeratin-fibre containing starting material, the reducing agent(s), andbuffer, and the pH, temperature and duration of solubilisation arepreferably chosen such that a satisfactory yield of solubilised keratinsis obtained, preferably at least 10, 20, 30, 40, 50 or 60% of thekeratin in the keratin-fibre containing starting material aresolubilised. The conditions of solubilisation are further preferablychosen such that the solubilised keratin is partially hydrolysed orpartially degraded to a degree further specified hereinbelow.

Preferably at least 10 g of keratin-fibre containing starting materialis solubilised per liter of (aqueous) solubilisation medium, andpreferably no more than 100 g of keratin-fibre containing startingmaterial is solubilised per liter of (aqueous) solubilisation medium.Usually between 20 and 60 g of keratin-fibre containing startingmaterial is solubilised per liter of (aqueous) solubilisation medium.

The concentration of the reducing agent in the (aqueous) solubilisationmedium, e.g. an alkali metalsulphide or ammonium sulphide is preferablybetween 0.05 M and 1.0 M. Alternatively, using the combination of2-mercaptoethanol and sodium hydroxide: 2-mercaptoethanol is used at aconcentration between 0.1 M and 1.5 M in combination sodium hydroxide atbetween 0.1 and 1.0 N.

The pH at which the keratins are solubilised is alkaline, i.e. higherthan pH 7.0 . Preferably however, solubilisation is performed at analkaline pH that is et least pH 8.0, 8.5, 9.0, 9.5, however, morepreferably the pH is at least or higher than pH 10.0, 10.5, 11.0, 11.5,12.0, or 12.5 because at a pH at least or higher than pH 10.0 thedissociation equilibrium of sulphide shifts towards S²⁻, which is astronger reductor than is HS⁻. Preferably the pH is not higher than pH13.5.

The temperature at which the keratins are solubilised preferably is atleast 20° C. However, preferably higher temperatures are used forsolubilisation, such as a temperature of preferably at least 30, 40, 50,60, 70 or 80° C., but preferably not higher than 100° C.

The duration of the keratin solubilisation step is primarily chosen suchthat the desired degree of hydrolysation of the solubilised keratin isobtained under the given solubilisation conditions. Typically thesolubilisation will take between 10 minutes and 24 hours. The durationof the keratin solubilisation may be further optimised for the yield ofsolubilisation. Thus the skilled person will empirically optimise theset of conditions for keratin solubilisation in order to obtained atleast the desired degree of keratin hydrolysis and preferably thehighest yield of solubilised keratin.

In addition to the solubilisation of the keratin, the process of theinvention further comprises the step of partially modifying the —SHgroups present in the keratin. Generally, these —SH groups will be free—SH groups that result from the cleavage of the intra- andintermolecular disulphide bonds (C) in the keratin molecules, as mayoccur during the solubilisation under reducing conditions of the keratinfrom the keratin-fibre containing starting material. Thus, usually, thefree —SH groups will be cysteine residues. The partial modification ofthe free —SH groups generally involves chemical conversion of the —SHgroup to another group, e.g. a functional group including but notlimited to one or more of the functional groups mentioned hereinbelow.One purpose of the modification is that it excludes the modified —SHgroups from (re)forming intra- and intermolecular disulphide bondsbetween the solubilised keratin molecules, thereby avoiding or at leastreducing the formation of insoluble keratin aggregates. On the otherhand partial modification of the —SH groups means that the remainingfree cysteine residues are available for further reaction, e.g.(re)formation of disulphide linkages, for crosslinking and/or forpolymerisation, which reactions may be used to impart desired propertiesupon the final product, such as mechanical strength and/or resistanceagainst chemical or physical influences. Some further advantages ofpartial modification—compared to essentially completemodification—include that the keratins thus obtained are (better) suitedto provide water-insoluble films, e.g. for applications in coatings andfor other applications mentioned hereinbelow. By comparison, fullymodified keratins, such as those described in U.S. Pat. No. 3,464,825mentioned above, are generally water-soluble. The partial modificationof the free —SH groups of the solubilised keratin may be achieved in anumber of manners known per se, which will mainly depend upon thefunctional group(s) used. By manner of non-limiting example, somesuitable functional groups as well as conditions for partiallyconverting the free —SH groups will be mentioned hereinbelow.

In the process of the invention the partial modification of thesolubilised keratin is preferably such that at least 10%, preferably atleast 20%, more preferably at least 30% and most preferably at least 40%of all free —SH groups in the solubilised keratin is modified, and atthe same time such that less than 70%, preferably no more than 65%, morepreferably no more than 60% and most preferably no more than 57% of allfree —SH groups in the solubilised keratin is modified. Ultimatelypreferred is a degree of modification of the all free —SH groups in thesolubilised keratin is about 50%, i.e. between 44 and 56%. The remaining—SH groups in the keratin will not be modified and thus will still bepresent as such in the partially modified product obtained, or asre-formed disulphide bonds (which will be considered equivalent to twofree —SH groups, e.g. for the purposes of calculating the degree ofmodification as described below).

The degree of modification may be controlled by suitably choosing theconditions of the modification reaction, including but not limited tothe functionalising agent used, the reaction time, the temperature, thepH, the solvent(s) used, the concentrations of the respective reactants,and the concentration of the keratin material. It is envisaged that theselection of the specific conditions that will lead to a final productwith the desired degree of modification may require some preliminaryexperiments and/or some degree of trial and error. However, based uponthe disclosure herein, this will be within easy reach of the skilledperson and should therefore be considered encompassed within the scopeof the invention.

The modification is carried out in a manner known per se, depending uponthe group or groups used to modify the —SH groups and whether thismodification is combined with any further processing steps, e.g. thesolubilisation. Suitable conditions may include the use of water or anaqueous medium at a concentration of the functionalising agent between0.01M and 1M, at a pH of between 7.0 and 10.0, at a temperature ofbetween 4° C. and 20° C., during a time of between 10 minutes and 24hours, and at concentration of solubilised keratin of between 2 g and 10g per 100 ml of the aqueous hydrolysis medium. Preferred conditions formodification with monochoroacetic acid at a degree of modification ofabout 50% are e.g. an incubation for 30–90 minutes of 10–30 gsolubilised keratin in a volume of 1 liter with 0.5–5 g monochloroaceticacid, at a pH between 8.5 and 9.5, and at a temperature between 10–20°C.

The degree of modification may be determined by comparing the amount offree —SH groups remaining after modification (including the amount ofdisulphide bonds present) with the amount of free —SH groups (includingdisulphide bonds) prior to modification, in which amount of free —SHgroups/disulphide bonds in a keratin preparation may be determined usinga suitable assay, such as the DTNB/NTSB assay as described by Schrooyenet al. (Journal of Agricultural and Food Chemistry; 2000; 48(9);4326–4334). Alternatively, the amount of free —SH groups and/disulphidebonds remaining after modification may also be compared to a theoreticalvalue for the amount of —SH groups/disulphide bonds in keratin. Thetheoretical amount of —SH groups/disulphide bonds in native keratin(based upon the amount of cystine/cysteine residues present) is about700 μmol cysteine groups/g keratin (with the amount of cystine groupsbeing half that amount).

The —SH group may be modified with any desired functional group suitedfor the functionalisation of —SH groups and/or for the functionalisationof keratins; or with a suitable combination of two or more of suchgroups. The specific functional group(s) chosen and the respectiveamounts thereof, will usually depend upon the desired properties of thefinal product.

Thus, the keratins of the invention may be provided with one or morenegatively charged functional groups; one or more positively chargedfunctional groups; and/or one or more neutral functional groups; or asuitable combination thereof. In this respect, it will be clear to theskilled person that a “positively” and/or “negatively” charged group maybe associated with a suitable counter-ion, i.e. an anion (i.e. group) orcation (i.e. group) respectively. It will also be clear that the actualcharge carried by a “positively” or “negatively” charged functionalgroup as described herein may also depend upon the conditions in whichthe keratins are present/maintained, such as the pH, the solvent, etc.Generally, however, at a pH in the range of 6.0 to 8.0 a “positively”charged group will have a net positive charge, a “negatively” chargedgroup will have a net negative charge, and a “neutral” group will have anet charge of essentially zero.

Preferably, the functional groups described will be introduced using oneor more saturated or unsaturated organic compounds that at least containa group or residue that allows the organic compound to react with an —SHgroup. These may include but are not limited to suitable leaving groupssuch as halogen (chloro, bromo or iodo-); epoxide- or glycidyl-groups orunsaturated groups such as (meth)acryl, vinyl; and other suitable groupswill be clear to the skilled person. Besides the group that may reactwith the free —SH groups, these organic compounds may be also containone or more further groups that may provide for a negative charge, suchas a —COOH group; or that may provide for a positive charge, such as anquaternary amine-group, including but not limited to alkylated aminegroups. Thus, for instance, the keratin may be modified with negativelycharged groups, e.g. those that can be introduced using for example:

-   halogenated acids such as chloroacetic acid; iodoacetic acid and    bromoacetic acid.-   peroxides such as hydrogen peroxide or organic peroxides, including    but not limited to performic acid or peracetic acid to form    sulphonate groups;-   unsaturated organic compounds that contain negatively charged    groups, such as vinyl compounds containing a —COOH group;-   glycidyl compounds;    or any suitable combination thereof

Usually, partially modifying a partially hydrolysed keratin with suchnegatively charged groups will enhance the dispersibility of thekeratins.

Positively charged functional groups may for example be introducedusing:

-   halogenated organic amines, such as halogenated alkylamines;-   glycidyl compounds carrying an amine group such as    glycidyltrimethylammoniumchloride;    or a suitable combination thereof.

Neutral functional groups may for instance be introduced using:

-   halogenated organic compounds such as halogenated alkanes and    halogenated ethers, esters or amides;-   glycidyl compounds such as alkylglycidyl compounds;-   vinylcompounds such as alkylvinylcompounds;    or a suitable combination thereof

Other suitable functionalising compounds are for instance described inFR 2 522 657. The keratins may also be modified using sulphites such ase.g. sodium sulphite or sodium metabisulphite to form S-sulfonategroups, or using other inorganic compounds.

It may be convenient to combine the modification step with thesolubilisation step, e.g. by adding the functionalising agent to thesolubilisation mixture. After solubilisation and modification thekeratin-derived product may already be suitable for its intended finaluse, and thus for instance may be marketed or otherwise provided as suchto the end-user.

In the process of the invention, the conditions of solubilisation and/ormodification are preferably such that the solubilised keratin ispartially hydrolysed or partially degraded. Partial hydrolysis—alsoreferred to as partial “degradation”—generally comprises cleavage of(part of) the peptide bonds between the amino acids that form thekeratin molecule. Partial hydrolysis may be achieved duringsolubilisation and/or modification. However, if solubilisation and/ormodification does not produce the desired degree of partial hydrolysisof the solubilised keratin, further hydrolysis or degradation may beachieved by any manner known per se in the art, including e.g. chemicalhydrolysis, physical hydrolysis and/or enzymatic hydrolysis.

The degree of hydrolysis of the solubilised keratin is such that akeratin-based product produced from the solubilised and partiallyhydrolysed keratin has the required physical and chemical properties.E.g. a film or coating produced from the solubilised and partiallyhydrolysed keratin preferably has a tensile strength higher than 15 MPa,more preferably higher than 16, 17, 18 or 20 MPa. The film preferablyalso has an E-modulus of at least 100 MPa, more preferably higher than150, 200, 250 or 300 MPa. The film preferably also has an elongation atbreak of at least 10%, more preferably higher than 20, 30, 40 or 50%. Adegree of hydrolysis of the solubilised keratin that allows to produce akeratin-based product with such physicochemical properties may bedefined by means of the distribution of the molecular weights of thesolubilised and partially hydrolysed keratin as obtained in the processof the invention. Thus, the distribution of the molecular weights of thesolubilised and partially hydrolysed keratin may be defined as follows:

-   The solubilised and partially hydrolysed keratin essentially has a    molecular weight of between 1 and 10.4 kDa, and in particular    between 3 and 10.4 kDa, whereby essentially is understood to mean    that at least 50%, preferably at least 90%, more preferably at least    95%, and most preferably at least 99% of all keratin molecules    present in the partially hydrolysed keratin fraction have a    molecular weight within the ranges indicated;-   Preferably at least 1% of the solubilised keratin has a molecular    weight less than 10 kDa In addition, preferably at least 50% of the    solubilised keratin has a molecular weight of more than 5 kDa. More    preferably, at least 3, 5, 7, 10 or 15% of the solubilised keratin    has a molecular weight less than 10 kDa, whereby at least 60, 70,    80, 90 or 95% of the solubilised keratin has a molecular weight of    more than 5 kDa; and/or-   The solubilised and partially hydrolysed the keratin preferably has    molecular weight distribution that is essentially equal to a    distribution of molecular weights of solubilised keratins that are    obtained when 40 grams of cleaned and dried poultry feathers are    solubilised in one liter of an aqueous solution of 0.05–0.5 M sodium    sulphide at a pH between pH 10.0 and pH 13.5, at a temperature    between 40 and 80° C. for 30–90 minutes. The solubilised keratins    are then preferably separated from the undissolved starting material    by filtration. More preferred, the molecular weight distribution is    essentially equal to a distribution of molecular weights of keratin    that is obtained when 40 grams of cleaned and dried poultry feathers    are solubilised in one liter of an aqueous solution of 0.075–0.15 M    sodium sulphide at a pH between pH 11.0 and pH 13.0, at a    temperature between 55 and 65° C. for 45–75 minutes. Most preferred,    the molecular weight distribution is essentially equal to a    distribution of molecular weights of keratin that is obtained when    40 grams of cleaned and dried poultry feathers are solubilised in    one liter of an aqueous solution of 0.1 M sodium sulphide at pH    12.5, at a temperature of 60° C. for 45–75 minutes.-   The solubilised and partially hydrolysed keratin preferably has    molecular weight distribution that is such that a 1% (w/v) solution    of the solubilised keratin at 25° has a viscosity of between 1 mPa.s    to 100 mPa.s, preferably between 1 mPa.s and 20 mPa.s. The viscosity    may for instance be measured in an Ubbelohde viscosimeter.-   The solubilised and partially hydrolysed the keratin preferably has    molecular weight distribution that the solubilised keratin has a    dispersibility in water of between 1% and 50%, preferably between    10% and 30%. The dispersibility of the keratin is herein defined in    that the keratin is capable of forming a stable dispersion which    shows little or no deposit within 24 hours after preparation.

The degree of hydrolysis may be controlled by suitably choosing theconditions of the hydrolysis reaction, including but not limited to thereactants used (e.g. for chemical degradation), the forces/equipmentused (e.g. for physical degradation), the enzymes used (e.g. forenzymatic degradation), the time of hydrolysis, the temperature, the pH,the solvent(s) used, the concentrations of the respective reactants, theconcentration of the keratin material, the manner in which the keratinmaterial is provided (e.g. after suitable pre-treatment as describedhereinbelow), any stirring or agitation used; and/or whether thehydrolysis is obtained during solubilisation, modification and/orseparately. It is envisaged that the selection of the specificconditions that will provide the desired degree of hydrolysis—and thatthereby, in conjunction with the partial modification and any furtherprocessing, lead to a final product with the desired properties, e.g.those described below—may require some preliminary experiments and/orsome degree of trial and error. However, based upon the disclosureherein, this will be within easy reach of the skilled person and shouldtherefore be considered encompassed within the scope of the invention.

In addition—or alternatively—the degree of hydrolysis may be determinedin several ways, including e.g. analysis of the molecular weights of thehydrolysed fragments or the distribution thereof, e.g. by size exclusionchromatography; determination of the viscosity of the preparation;determination of dispersibility of the preparation; determination of theamount of protein end-groups; or any combination of these and othersuitable techniques.

Depending upon the conditions used, a fraction or preparation ofpartially hydrolysed keratins with molecular weights that areessentially within the ranges indicated above may be obtained directlyas a result of the partial hydrolysis. However, it is also within thescope of the invention that, in order to provide such a fraction orpreparation, a certain amount of—and up to essentially all—highermolecular weight components still present (e.g. any remaining andundissolved starting material) and/or a certain amount of—and up toessentially all—lower molecular weight degradation products formedduring the hydrolysis reaction, may be removed from the hydrolysedfraction or preparation, e.g. as part of any further processing afterhydrolysis and/or modification.

Also, according to the invention, it is possible that the solubilisedkeratin may form aggregates, e.g. with molecular weights above therange(s) mentioned above. Such aggregation as well as the aggregatesobtained are within the scope of the invention, as long as thekeratin-derived molecules that form these aggregates have molecularweights that are essentially within the range(s) indicated.

Furthermore, it is also possible that some degree of polymerisation ofthe keratin-derived products takes place (e.g. after partial hydrolysis,partial modification and/or further processing), in which again productswith molecular weights above the range(s) mentioned above may be formed.Such polymerisation as well as the polymeric products obtained are alsowithin the scope of the invention, again provided that thekeratin-derived molecules that combine to form these polymericstructures have molecular weights that are essentially within therange(s) mentioned above.

Compared to the essentially intact keratins of the prior art, thepartially hydrolysed/modified keratins of the invention mayadvantageously be used in particularly those applications where keratinswith a more hydrophobic character and/or a lower surface tension isrequired. This may be advantageous when the partially hydrolysedkeratins of the invention are applied in films or coating. The partiallyhydrolysed keratins of the invention also have improved adhesiveproperties as compared to essentially intact keratins. These adhesiveproperties may e.g. be advantageously applied in sticking paper to glassas may be used in fixing labels onto (beer) bottles. Compared to theessentially intact keratins of the prior art, the partially hydrolysedkeratins of the invention have improved emulgating properties as thepartially hydrolysed keratins are more amphiphilic. A further benefit ofusing partially hydrolysed and partially modified keratins lies in thepotentially increased antimicrobial effect of partially hydrolysedproteins. This was demonstrated for lactoferricin which is a hydrolysisproduct of the whey protein lactoferrin. By partial modification, thekeratin peptide with increased antimicrobial effect can still beincorporated into a film with good mechanical strength by disulphidebond formation, leading to a new generation of anti-microbial coatings.

In a further optional aspect of the invention, the solubilised andpartially modified keratin may be subjected to further processing steps,which may e.g. include:

-   further purification, e.g. to remove undesired components and/or    substances from the keratin product obtained These may for instance    include remaining keratin starting material, higher molecular weight    keratin components, lower molecular weight keratin components,    reactants and/or by-products from any of the processing steps,    and/or other impurities or undesired components. Suitable techniques    for removing such components or substances include e.g. washing,    precipitation, dialysis, filtration and centrifugation;-   isolation of the keratin-derived product, and/or of any specific    part or fraction thereof. This may for instance be achieved using    techniques such as precipitation, membrane separation,    chromatographic techniques, and solvent extraction;-   drying, including e.g. freeze-drying, spray-drying, multi-stage    drying and drying using a roll;-   dispersion in a desired solvent or mixture of solvents;-   aggregation or even polymerisation;    or any suitable combination thereof.

Also, depending upon the intended use, the keratin product may becombined or mixed with one or more further substances, additives,components, etc. Some non limiting examples thereof include pigments,salts, anti-microbial agents, detergents, and plasticizers.

It will be clear that any such further processing and/or addition ofafter components may also be carried out by the end-user; and this isalso encompassed within the scope of the invention.

A further aspect of the invention relates to compositions comprising thekeratins obtained or obtainable by a process according to the inventionas described above. The keratin in these compositions is characterisedin that (a) its —SH groups are partially modified, preferably at least10% and no more than 70% of the —SH groups of the keratin are modified;and (b) it is partially hydrolysed such that the keratins in thecomposition have a molecular weight distribution as specified hereinabove.

In another aspect, the invention discloses compositions comprisingkeratin, which compositions are obtainable (or obtained) in a processcomprising at least the steps of (a) solubilising keratin from akeratin-fibre containing starting material in an aqueous solution of0.05–0.5 M sodium sulphide at a pH between pH 10.0 and pH 13.5, at atemperature between 40 and 80° C. for 30–90 minutes; and (b) modifyingbetween 10 and 70% of the —SH groups of the solubilised keratin,preferably by alkylation.

Generally, the product of the invention can be described as a keratinous(e.g. keratin-based or keratin-derived) product or a preparation orcomposition wherein partially hydrolysed and partially modified keratinmolecules form the major component, e.g. for at least 50 wt. %,preferably at least 80, 90, 95 or 99 wt. %, of the total components inthe product, preparation or composition. Preferably the partiallyhydrolysed and partially modified keratin molecules form at least 50 wt.%, preferably at least 80, 90, 95 or 99 wt. %, of the total protein inthe product, preparation or composition. Thus, it is not excluded thatthe proteinaceous product of the invention may contain some othercomponents, such as other proteins or protein constituents (e.g.hydrolysed and/or modified proteins), non-hydrolysed keratins, low(er)molecular weight hydrolysis products, as well as non-protein componentssuch as surfactants, salts, and/or impurities such as dirt. However,these will only be present in minor amounts, e.g. of less than 50 wt. %,preferably less than 20 wt. %, more preferably less than 10 wt. %, evenmore preferably less than 5 wt. %.

Preferably at least 50, 60, 70, 80, or 90% of the keratin molecules inthe compositions of the invention comprise at least one hydrophobic partor region and at least one, and preferably two hydrophilic parts orregions, as further described below. E.g., these keratin molecules maycomprise a total of between 10 and 100 amino acid residues, preferablybetween 30 and 100 amino acid residues; in which the hydrophobic part orregion comprises between 20 and 40 amino acid residues, the remainingamino acid residues constituting the hydrophilic part(s) or region(s).In a preferred aspect of the invention, the keratins in the compositionare hydrolysed to such an extent that the resulting partially hydrolysedkeratin molecules (still) contain at least a hydrophobic part orregion—e.g. derived from the original central part (β) of the originalkeratin molecule—and at least one hydrophilic part or region, e.g.derived from one of the original chain ends (C) and/or (N). Such apartially hydrolysed keratin molecule can be considered to have theschematic structure “A-B” in which A represents the hydrophilic part orregion, generally of between 5 to 30 amino acid residues; and Brepresents the hydrophobic part or region, generally of between 5 and 40amino acid residues. Even more preferably, in the invention, thekeratins are hydrolysed to such an extent that the resulting partiallyhydrolysed keratin molecules (still) contain at least a hydrophobic partor region—e.g. derived from the original central part (β) of theoriginal keratin molecule—flanked on both ends by a hydrophilic part orregion, e.g. one derived from the original chain end (C) and one derivedfrom the original chain end (N). In this preferred aspect, the partiallyhydrolysed keratin molecule can be considered to have the schematicstructure “A-B-A” in which both A's represents the hydrophilic parts orregions (e.g. as defined above) and B represents the hydrophobic part orregion (also as defined above).

Because of this preferred structure A-B or even more preferred structureA-B-A, the partially hydrolysed keratins of the invention may be usedwith advantage to prepare or provide multi-phasic system—such asdispersions, emulsions, gels, micelles, microspheres—as well as layersor layered structures (including but not limited to single or doublelayers). Also, the partially hydrolysed keratins of the invention may beused as stabilisers, emulsifying agents or more generally formulatingagents for such multi-phasic systems, which may be aqueous systems ororganic systems. It will be clear to the skilled person that thewater-soluble keratins or keratin-preparations described in the art,including but not limited to (more) fully hydrolysed preparations, willgenerally not be suited for such applications.

The compositions according to the invention will often be in the form ofan aqueous solution or dispersion of the partially modified andpartially hydrolysed keratins. Such solutions or dispersions willpreferably contain at least 10, 20, or 40 g of keratins per liter andpreferably no more than 60, 75, or 90 g of keratins per liter. Generallysuch solution or dispersions will contain about 50 g of keratins perliter. Preferably these solution or dispersion are stable in both achemical and physical sense. Thus, in a chemical sense, no appreciable,i.e. preferably less than 10, 5, 1%, (further) degradation, modificationand/or oxidation of the keratins occurs over a period of preferably atleast a day, a week or a month. In a physical sense, no appreciable,i.e. preferably less than 10, 5, 1%, sedimentation or precipitation ofthe keratins occurs over a period of preferably at least a day, a weekor a month. In order to stabilise the solutions or dispersions of theinvention, additives known in the art per se may be applied. Forlonger-term storage or for more convenient transportation thecompositions of the invention may be in a solid form, preferably in theform of a dispersible non-dusting powder or granulate. The usualtechniques for drying and/or granulating may be applied, including theuse of additives to aid in the formulation of the solid form.

A further aspect of the invention relates to a process for producing akeratin-based product using the partially modified and partiallyhydrolysed keratins of the invention as source of keratin. Preferredkeratin-based products of the invention are produced by casting asolution or dispersion of the keratins of the invention.

Another aspect of the invention concerns the keratin-based productproduced from the partially modified and partially hydrolysed keratinsof the invention. In a preferred embodiment, the keratin-based productis a film or a coating casted from a solution or dispersion of thekeratins of the inventions. Preferably, the film or a coating has atensile strength higher than 15 MPa. The film or a coating preferablyhas an E-modulus higher than 100 MPa and preferably an elongation atbreak of more than 10%.

The partially modified and partially hydrolysed keratins of theinvention may be used in any application for keratin-based productsknown in the art, including e.g. those applications mentioned in theprior art given hereinabove. Generally, in these applications, thekeratin-derived products of the invention will provide favourableproperties including but not limited to improved mechanical propertiessuch as mechanical stability; improved physical and chemical stability;low solubility in water and good film-forming properties. In addition,because the keratin-derived product of the invention iswater-dispersible instead of water-soluble, it may also be used in (thepreparation of) for instance dispersions, emulsions, micelles, gels,microspheres or other multi-phasic aqueous systems. Thus, somenon-limiting uses of the keratins of the invention include:

-   use as or in films, coatings, etc., or in the preparation thereof;-   use as or in (biodegradable) packaging materials, or in the    production thereof;-   use as or in formulations such as controlled release systems, e.g.    for active substances such as pharmaceuticals; agrochemicals such as    herbicides, pesticides or other biocides; flavorings; perfumes;    etc.;-   use as or in the formation of emulsions, dispersions or other    multi-phasic aqueous systems;-   use as or in fillers, gelating agents, binders, bulking agents,    granulating agents, release agents, matrix materials, emulsifiers,    stabilisers or other formulating agents;-   use as antioxidants;-   use as anti-microbial agents.

As such, the keratins of the invention may for example find use in foodproducts or in the field of food technology generally; in pharmaceuticaland veterinary products; in cosmetics; in the field of agrochemicals; inadhesives; in paints or other coatings; in packaging materials; incleansing agents such as detergents; in agriculture. Some specific usesof the keratins of the invention that are envisaged include, but are notlimited to:

-   use as coating or binders for granules, powders etc. such as washing    powder or other detergents;-   use in general purpose adhesives, both for industrial as well as    household use;-   use in binder or adhesive for wood, paper, paperboard or moulded    fibre;-   use as binders in coatings—including but not limited to water-borne    paint systems—and/or inks/ink systems; both for industrial as well    as household use;-   use as anti-oxidant;-   use in encapsulating and/or coating technology;-   use as anti-microbial agents in for instance animal feed and    cosmetics.

DESCRIPTION OF THE FIGURES

FIG. 1: Schematic representation of (A) a keratin molecule, (B) amicrofibril consisting of polymerized keratin units and (C) the inter-and intra-molecular disulphide bonds in a microfibril.

FIG. 2: A plot of the molar mass of three different modified keratinpreparations versus the elution volume on a size exclusion column.

FIG. 3: Solubility of Unmodified Keratin in various solutions: (1) inwater; (2) in 50 mM Tris-buffer pH 8.0, (3) in 8M urea at pH 8.5, and(4) in 8M urea at pH 8.5 supplemented with 2.5 g dithiothreitol (DTT)per liter.

FIG. 4: Solubility of 50%-Modified Keratin in various solutions: (1) inwater; (2) in 50 mM Tris-buffer pH 8.0, (3) in 8M urea at pH 8.5, and(4) in 8M urea at pH 8.5 supplemented with 2.5 g dithiotreitol (DTT) perliter.

FIG. 5: Solubility of 90%-Modified Keratin in various solutions; (1) inwater; (2) in 50 mM Tris-buffer pH 8.0, (3) in 8M urea at pH 8.5, and(4) in 8M urea at pH 8.5 supplemented with 2.5 g dithiothreitol (DTT)per liter.

EXAMPLES Example 1 Preparation of Partially Modified DispersibleKeratins with and without Partial Degradation

In one comparative experiment the procedure described by Schrooyen etal. (Journal of Agricultural and Food Chemistry; 2000; 48(9); 4326–4334)is used. Poultry feathers were cleaned using water and detergents.Cleaned and dried feathers (40 g) were mixed with one liter of anaqueous solution of 2-mercaptoethanol (125 mM), urea (8M) and EDTA (3mM) in Tris-buffer (0.2 M, pH 9.0) and stirred for 1 hour. Undissolvedfeathers were separated from the dissolved keratins using a cheese clothand a Whatman 54 filter (10 μm pore size). After filtration, 2 grams ofmonochloroacetic acid was added to the filtrate and the pH was keptconstant at 9.0. After 1 hour the aqueous solution was dialysed andlyophilised. The yield of dry keratin product was 45%, based on 100%keratin starting material (weight of feathers). This product will bereferred to as Modified Keratin—Method 1.

In a second experiment, a modified method, Method 2 was used. Incomparison to Method 1, Method 2 differs in following aspects:

-   1) no urea is used-   2) the pH is more alkaline (12.5 instead of 9.0)-   3) a higher temperature is used (60° C. instead of 20° C.)

These conditions lead to partial degradation of the keratin proteins byhydrolysis. Cleaned and dried feathers (40 g) were mixed with one literof a hot aqueous Na₂S-solution (0.1M, pH 12.5, 60° C.) and stirred for 1hour. Undissolved feathers were separated from the dissolved keratins acheese cloth and a Whatman 54 filter (10 μm pore size). After cooling to20° C., 2 grams of monochloroacetic acid was added to the filtrate andthe pH was set at 9.0 (yielding essentially 50% of SH-modification,referred to as 50%-Modified Keratin). After 1 hour the keratins wereprecipitated by setting the pH at 4.2 using hydrochloric acid (2N). Theprecipitates were isolated by centrifugation in a Sorvall centrifuge at20,000×g for 30 minutes. A sample was taken from the supernatant andlyophilised for further analysis (further referred to as ModifiedKeratin Supenatant—Method 2). Keratin pellets were washed with aceticacid (0.1N, pH 4.2) and subsequently resuspended in water. The pH of theresuspended pellets was set at 7.0 using NaOH (1N) and these mixtureswere freeze dried. The yield of dry keratin product was 40%, based on100% keratin starting material (weight of feathers). This product willbe referred to as Modified Keratin—Method 2.

Degree of Modification. The degree of modification was measured usingDTNB/NTSB-assay as described by Schrooyen et al. (Journal ofAgricultural and Food Chemistry; 2000; 48(9); 4326–4334). For ModifiedKeratin—Method 1, 54% of the cysteine residues were modified, forModified Keratin—Method 2, 57% of the cysteine residues were modified.

In FIG. 2, a plot of the molar mass versus the elation volume (TSK G3000+TSK Guard PWH column+UV detector) is shown for ModifiedKeratin—Method 1, Modified Keratin Supernatant—Method 2 and ModifiedKeratin—Method 2. The eluent was an aqueous solution of urea (8M) anddithiothreitol (15 mM). The flow rate was 0.7 ml/min. Under these eluentconditions all remaining disulphide bonds are reduced and only thekeratins (≈10,400 g/mol) or degradation products (less than 10,400g/mol) are observed. It can be seen that both Modified Keratin—Method 1and Modified Keratin—Method 2 have a similar molar mass distribution,although Modified Keratin—Method 2 contains more high and low molar massproducts than Modified Keratin—Method 1. The high molar mass productsare possibly products of cross-linking reactions which can occur at highpH, e.g. by lanthionine formation. Low molar mass products are theresult of degradation. This can be observed even better in ModifiedKeratin Supernatant—Method 2, which still contains a large amount of lowmolar mass products, mainly between 3,000 and 10,000 g/mol.

As the pH used in Method 1 is never higher than 9.0, degradation of thepolypeptide chain is unlikely to occur, as confirmed using SEC-MALLSanalysis. In Method 2 the high temperature and strongly alkaline pHcause partial degradation of the keratins.

Example 2 Preparation of Partially Degraded Keratins with VariousDegrees of Modification

Poultry feathers were cleaned using water and detergents. Cleaned anddried feathers (60 g) were mixed with 1.5 liters of a hot aqueous(NH₄)₂S-solution (0.1M, pH 12.5, 60° C.) and stirred for 1 hour.Undissolved feathers were separated from the dissolved keratins using acheese cloth and a Whatman 54 filter (10 μm pore size). The keratinyield in the filtrate was 59.5%, based on 100% keratin starting material(feathers). After cooling to 20° C., the filtrate was split in 3 partsof 500 ml each: to one part no monochloroacetic acid was added and thepH was set at 9.0 (yielding essentially unmodified keratin, referred toas Unmodified Keratin), to a second part 1 gram of monochloroacetic acidwas added and the pH was set at 9.0 (yielding essentially 50% ofSH-modification, refereed to as 50%-Modified Keratin), to a third part 5grams of monochloroacetic acid was added and the pH was set at 9.0(yielding essentially 90% of SH-modification, referred to as90%-Modified Keratin). After 1 hour the keratins were precipitated bysetting the pH at 4.2 using hydrochloric acid (2N). The precipitateswere isolated by centrifugation in a Sorvall centrifuge at 20,000×g for30 minutes. A sample was taken from the supernatant for analysis.Keratin pellets were washed with acetic acid (0.1N, pH 4.2) andsubsequently resuspended in water. The pH of the resuspended pellets wasset at 7.0 using NaOH (1N) and these mixtures were freeze dried. Theyield of dry keratin product was 46.5% for Unmodified Keratin, 48.9% for50%-Modified Keratin and 20.8% for 90%-Modified Keratin, based on 100%keratin starting material (weight of feathers).

Degree of Modification. The degree of modification was measured usingDTNB/NTSB-assay as described by Schrooyen et al. (Journal ofAgricultural and Food Chemistry; 2000; 48(9); 4326–4334), For UnmodifiedKeratin 1, 0% of the cysteine residues were modified, for 50% ModifiedKeratin 53% of the cysteine residues were modified, for 90%-ModifiedKeratin, 89% of the cysteine residues were modified.

Solubility

The freeze dried keratin products were suspended (5%) in 4 buffers atroom temperature:

-   1. Water-   2. Aqueous Tris-buffer (50 mM)—pH 8.0: an alkaline pH helps to    suspend keratins-   3. Urea (8M)−pH 8.5: urea is added to prevent aggregation of    non-covalently bound keratins-   4. Urea (8M)+2.5 g, ltr Dithiothreitol (DTT)—pH 8.5: DTT is added to    reduce disulphide bonds

The Unmodified Keratin is completely soluble in Buffer 4 (FIG. 3). Itforms a white gel in the other buffers. The 50%-Modified Keratin issoluble in Buffer 3 and 4 (FIG. 4). It forms a white gel in the otherbuffers. When buffer 2 is heated to 40° C. a turbid dispersion isobtained. The 90% -Modified Keratin is soluble or dispersible in allbuffers; Buffer 1 and 2 remain somewhat turbid (FIG. 5).

Film Formation

To produce films by solution casting, a good dispersion or solution inwater is necessary. From Unmodified Keratin it was not possible toproduce films. From the 50%-Modified Keratin a 5%-dispersion was mixedwith glycerol (0.30 g/g keratin) and cast in a petri dish. After drying,a strong film (tensile strength 15 MPa) was obtained, which was notsoluble in water at room temperature. From the 90%-Modified Keratin asimilar film was prepared. This film had bad mechanical properties(tensile strength<5 MPa) and was water soluble.

Adhesive Properties

The 50%-Modified Keratin had good adhesive properties and was especiallysuitable for sticking paper to glass, which is often used for beerbottles.

1. A process for producing partially modified and partially hydrolysedkeratin, the process comprises the steps of (a) solubilising keratinfrom a keratin-fibre containing starting material in an aqueous solutionusing a reducing agent at an alkaline pH of between pH 10 and pH 13.5and at a temperature of at least 40° C., and (b) partially modifying the—SH groups of the solubilised keratin by alkylation, characterised inthat the solubilised keratin is partially hydrolysed as a result of theconditions of steps (a) and (b) and an optional further hydrolysis step(c), whereby the solubilised keratin is partially hydrolysed such thatat least 50% of the solubilised keratin molecules has a molecular weighthigher than 1 kDa and less than 10.4 kDa and whereby the solubilisedkeratin has molecular weight distribution that is equal to adistribution of molecular weights of keratin that is obtained when 40grams of cleaned and dried poultry feathers are solubilised in one literof an aqueous solution of 0.05–0.5 M sodium sulphide at a pH between pH10.0 and pH 13.5, at a temperature between 40 and 80° C. for 30–90minutes.
 2. The process according to claim 1, whereby the solubilisedkeratin is partially hydrolysed such that at least 1% of the solubilisedkeratin has a molecular weight less than 10 kDa, and at least 50% of thesolubilised keratin has a molecular weight of more than 5 kDa.
 3. Theprocess according to claim 1, whereby at least 10% and no more than 70%of the —SH groups of the solubilised keratin are modified.
 4. Theprocess according to claim 1, whereby the reducing agent comprises asulphide, preferably an alkali metal sulphide or ammoniumsulphide.
 5. Acomposition comprising keratin, whereby the keratin is characterised inthat: a) at least 10% and no more than 70% of the —SH groups of thekeratin are modified by alkylation; and b) at least 50% solubilisedkeratin has a molecular weight of between 1 and 11 kDa and whereby thesolubilised keratin has molecular weight distribution that is equal to adistribution of molecular weights of keratin that is obtained when 40grams of cleaned and dried poultry feathers are solubilised in one literof an aqueous solution of 0.05–0.5 M sodium sulphide at a pH between pH10.0 and pH 13.5, at a temperature between 40 and 80° C. for 30–90minutes.
 6. The composition according to claim 5, whereby at least 1% ofthe keratin has a molecular weight less than 10 kDa, and at least 50% ofthe keratin has a molecular weight of more than 5 kDa.
 7. A compositioncomprising keratin, whereby the composition is obtained in a processcomprising the steps of: a) solubilising keratin from a keratin-fibrecontaining starting material in an aqueous solution of 0.05–0.5 M sodiumsulphide at a pH between pH 10.0 and pH 13.5, at a temperature between40 and 80° C. for 30–90 minutes; b) modifying between 10 and 70% of the—SH groups of the solubilised keratinby alkylation.
 8. The compositionaccording to claim 5, whereby the composition is in the form of anaqueous solution or dispersion.
 9. The composition according to claim 5,whereby the composition is in a solid form, preferably in the form of adispersible non-dusting powder or granulate.
 10. A process for producinga keratin-based product, comprising adding a keratin containingcomposition as defined in claim 5 as a source of keratin.
 11. Theprocess according to claim 10, wherein the keratin-based product isproduced by casting a solution or dispersion of said keratin containingcomposition.
 12. The process of claim 11, wherein said process producesa keratin-based product that is a film or a coating.
 13. A keratin-basedproduct produced from a keratin containing composition as defined inclaim 5, the product being a film or a coating with: a) a tensilestrength of higher than 15 MPa; b) an E-modulus higher than 100 MPa; andc) an elongation at break higher than 10%.
 14. A method for thepreparation of a material selected from the group consisting of films orcoatings; biodegradable packaging materials; formulations for controlledrelease systems for active substances; emulsions, dispersions ormultiphasic aqueous systems; fillers, gelling agents, binders, bulkingagents, granulating agents, release agents, matrix materials,emulsifiers, or stabilisers; an anti-oxidant; and an anti-microbialagent comprising adding the keratin as defined in claim 5 to saidmaterial.
 15. A biodegradable packaging material comprising thecomposition according to claim
 5. 16. A formulation for controlledrelease of an active substance, comprising the composition according toclaim
 5. 17. An emulsion, dispersion or multiphasic aqueous systemcomprising the composition according to claim
 5. 18. A filler, gellingagent, binder, bulking agent, granulating agent, release agent matrixmaterial, emulsifier or stabilizer, comprising the composition accordingto claim
 5. 19. An anti-oxidant comprising the composition according toclaim
 5. 20. An anti-microbial agent comprising the compositionaccording to claim 5.